FK506 is an immunosuppressant that inhibits T-cell activation and proliferation (B. E. Bierer et al., Current Opinions in Immunology 5, pp. 763–773 (1993)]. Immunosuppressants, such as FK506, are useful drugs in the treatment of transplant rejection and the prevention of autoimmune diseases. Furthermore, such compounds are useful tools in immune system research.
FK506 is a more recently discovered and more potent immunosuppressant than cyclosporin. Unfortunately, FK506 is characterized by undesirable pharmacological properties, such as toxicity and poor bioavailability [P. Neuhaus et al., Lancet, 344, pp. 423–428 (1994)]. Therefore, there remains a need for potent immunosuppressants with improved pharmacological properties.
FK506 acts as an immunosuppressant by inhibiting T-cell signal transduction pathways that control lymphokine transcription factors. As a result, gene activation of various lymphokines, including IL-2, is prevented. This in turn leads to an inhibition of T-cells, and therefore, immunosuppression.
FK506 exerts these effects in a step-wise process. Initially, FK506 binds to a peptidyl prolyl isomerase, FK506 Binding Protein (“FKBP12”). This complex then binds to, and inhibits, calcineurin. Subsequent events inhibit signal transduction pathways, inhibit lymphokine gene transcription, and ultimately, reduce production of lymphokines, such as IL-2.
Calcineurin is a Ca2+ L-dependent serine/threonine phosphatase. It is a heterodimer composed of 2 subunits: calcineurin A (“CnA”), a 59 kDa catalytic subunit and calcineurin B (“CnB”), a 19 kDa subunit. CnA contains a phosphatase active site and an autoinhibitory region as well as binding sites for calmodulin and CnB. Binding of FKBP12/FK506 inhibits the phosphatase activity of calcineurin against physiological substrates. FKBP12/FK506 does not, however, bind at the phosphatase active site.
Thus, a compound may inhibit calcineurin by binding to the phosphatase active site (“active site”), by binding to an accessory binding site, such as the FKBP12/FK506 binding site, or by binding to both sites simultaneously. Such compounds may interact directly with calcineurin or, alternatively, may bind to FKBP12, or a FKBP12 homologue, prior to binding to calcineurin.
FKBP12 has been characterized by its cDNA and amino acid sequences. The crystal structures of FKBP12, and of FKBP12 bound to FK506, have been reported. However, this structural information has not proven useful in the design of calcineurin inhibitors [M. V. Caffrey et al., Bioorg. Med. Chem. Lett., 21, pp. 2507–2510 (1994)].
Rat calcineurin has been characterized by its amino acid sequences and its cDNA. Human calcineurin has been characterized by its amino acid sequences and its cDNA [Guerini et al., Proc. Natl. Acad. Sci. USA, 86, pp. 9183–87 (1989)]. Knowledge of the primary structure, i.e., amino acid sequence, of calcineurin, however, does not allow prediction of its tertiary structure. Nor does it afford an understanding of the structural, conformational, and chemical interactions of calcineurin with FKBP12/FK506 or other compounds or inhibitors.
The crystal structure of calcineurin has not been reported. Nor has the crystal structure of a calcineurin homologue or a calcineurin co-complex been reported. The need, therefore, exists for determining the crystal structure of calcineurin to provide a more accurate description of the structure of calcineurin to aid in the design of improved inhibitors of calcineurin activity. The crystal structure of a complex comprising calcineurin A, calcineurin B, FKBP12, and FK506 would provide such a description.
Calcineurin inhibitors, such as FK506, have therapeutic potential as immunosuppressants. Specifically, such compounds may be used in the treatment of transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease, and other autoimmune diseases.